Hospital-acquired infections significantly increase the cost and morbidity of critically ill and critically injured patients. Many of the inflammatory and infection complications appear to be caused by overgrowth and increased virulence of bacteria within the patient's intestine. Two immunologic systems - acquired and the innate immunity - provide immune protection against bacteria to control them. Route and type of nutrition influence both systems. This laboratory defined defects in acquired immune defenses when the gut is not fed during parenteral nutrition (PN). Recently, we discovered defects in innate mucosal defenses and tracked these defects to failure to stimulate the enteric nervous system (ENS). We showed that providing ENS neuropeptides maintains aspects of both acquired and the innate immunity. There are 4 specific aims: Specific Aim 1: To determine whether increasing ENT stimulation or the administration of the neuropeptide, bombesin (BBS), in PN-fed mice increases intra-luminal and intra-Paneth cell innate immune molecules including lysozyme and defensin levels compared with PN feeding alone. The Paneth cells are the source of an important innate immune molecule, secretory phospholipase A2 (sPLA2). Our data show 1) a decrease in number and size of Paneth cell granules and sPLA2 in PN-fed mice compared with chow-fed mice and 2) that BBS given to PN-fed mice increases size of these granules. We hypothesize that ENS controls innate immunity. We will vary route of nutrition and treat PN-fed mice with BBS to confirm our data. Since Paneth granules contain other molecules besides sPLA2 which play a role in innate immunity, we will also investigate the effects of neuropeptides on lysozymes and defensins in addition to sPLA2. Specific Aim 2: To determine the effect of BBS with and without cholinergic stimulation on the bactericidal function of small intestinal fluids in PN-fed mice. Our preliminary data show that BBS increases the size of Paneth cell granules but does not increase the depressed sPLA2 gut levels in PN-fed mice. Our preliminary data show that cholinergic stimulation is required for granule release from Paneth cells. To test our hypothesis that both neuropeptides (for granule production) and cholinergic stimulation (for release) are necessary, we will determine the effect of neuropeptides with and without cholinergic stimulation on release of innate immune molecules and will determine their bactericidal potency in vitro in PN-fed mice. Specific Aim 3: To determine the effect of PN with or without BBS stimulation and with and without bethanecol stimulation on invasion of intestinal epithelial cells in culture by a virulent bacterial pathogen. Other investigators showed that stress, PN, and other factors stimulate gut bacteria to increase virulence and attack host tissues. We will investigate the ability of neuropeptides and cholinergic stimulation to prevent development of bacterial pathogen virulence using a model that studies the invasion of intestinal epithelial cells in culture with E. coli. Specific Aim 4: To examine th effect of BBS on acquired immunity (GALT cell populations & sIgA); innate immunity (sPLA2, lysozymes, bactericidal function of luminal secretions, and invasion of epithelial cells in culture by a virulent pathogen); bacterial colonization; and development of colitis in knockout mice lacking an ENS. To confirm validity of Specific Aims 1 & 3, this work will examine the use of neuropeptides in an ENS-deficient animal to determine if administering ENS products eliminates or palliates lethal colitis that develops. The proposed studies will provide mechanistic details about the role of the ENS in innate mucosal defenses and will identify novel agents to maintain normal gut homeostasis in stressed animals that are not fed enterally. We expect these studies will provide advances in our understanding of mucosal defenses and be highly relevant to critically injured and critically ill Veterans. PUBLIC HEALTH RELEVANCE: Enteral feeding improves outcome and reduces sepsis in critically ill and critically injured patients. Lack of feeding via the gastrointestinal tract causes significant deterioration in acquird immunity by reducing production of specific antibacterial immunoglobulins. Our preliminary studies show that antibacterial molecules of the innate immune system are also adversely affected, but administration of products of the enteric nervous system is capable of reversing these defects. Discoveries made during this research may form the basis for novel therapies to treat patients who cannot be fed via the gastrointestinal tract during their critical illness. Such therapies will maintain normal immunologic homeostasis that is typically driven by enteral feeding.